Our goal is to understand the biophysical mechanisms that control cell movement. Directed cell motility is essential for development, axon guidance, immune response, wound healing, and metastatic cancer. Understanding how to modulate this basic process will aid in the treatment of a host of diseases. External signals are processed by a guidance system that subsequently engages the actin polymerization machinery to drive local membrane protrusion. We focus on the signaling proteins that make up this guidance system. Most of these signaling molecules display complex allosteric regulation. For example, the neuronal Wiskott- Aldrich Syndrome Protein (N-WASP) stimulates actin filament nucleation by the Actin-related protein (Arp) 2/3 complex, but only when activated by the proper combination of upstream signaling inputs. N-WASP is also the point of attack by several bacterial pathogens that hijack the host cytoskeleton. Thus N-WASP and related proteins are critical control nodes in motility. Previously, we have studied how N-WASP functions as an complex allosteric switch. Our current aims are to: 1) Elucidate how pathogen proteins subvert N-WASP activity. We will study the structure and interactions of two pathogenic activators: the Shigella flexneri protein Ics A and the Enterohemorrhagic E. coli (EHEC) protein EspFu. 2) Elucidate the mechanism of allosteric control of guanine nucleotide exchange factors (GEFs) upstream of N-WASP. N-WASP and the related molecules are activated by Rho family GTPases, which are in turn controlled by specific GEFs. We will focus on the mechanism of regulation of two GEFs: Intersectin, which plays a key role in actin-mediated endocytosis;and P-Rex1, which plays a key role in neutrophil chemotaxis. 3) Engineer signaling switches that control actin polymerization in vivo. Our previous work suggests that protein domains can be used as building blocks to assemble signaling proteins with a variety of allosteric input/output behaviors. We have used this strategy to reprogram input control of N-WASP. We will extend these studies by attempting to reprogram other signaling molecules (specifically as GEF's) and testing if such engineered switches can be used to control actin polymerization in vivo and as tools to systematically dissect and perturb guidance circuits.